This invention relates to hematology systems and methods. More specifically, this invention relates to systems and methods for analyzing blood samples to identify, classify, and/or quantify nucleated red blood cells (nRBCs) in a sample of blood.
Nucleated red blood cells are often present in the fetus and in the peripheral blood of newborns. However, nRBCs are considered to be abnormal for adults. The presence of nRBCs in an adult's peripheral blood stream is usually an indication of serious marrow stress. Studies have shown that the appearance of nRBCs in the blood stream is highly correlated with severe disease stages and/or poor prognosis for critically ill patients. Therefore, accurate identification and quantification of nRBCs has become increasingly important for clinical diagnostics.
Because nRBCs share numerous similarities with white blood cells (WBCs), the concentration of nRBCs in a blood sample is typically reported as a percentage of total WBCs in the blood sample (i.e., % nRBC=nRBCs/WBCs×100%). Traditional approaches to analyze nRBCs include: (1) separating nRBCs from WBCs by size; (2) differentiating nRBCs from WBCs by means of light scattering; or (3) analyzing nRBCs by means of fluorescence emission detection after lysis and staining with a cell membrane impermeable fluorescent dye(s).
Each of the above-listed techniques has shown weaknesses in clinical practices. For example, it is difficult to completely eliminate fragments of lysed red blood cells (RBCs) in rapid hematology measurements. Because fragments of RBCs and the nuclei of nRBCs may be similar in size and light scattering characteristics, analysis based on size and/or light scattering is sometimes misleading. Meanwhile, analysis based on fluorescence emission may be adversely affected by: (1) “under-lysing” of the sample such that the cell membrane impermeable dye cannot reach the nuclei of the nRBCs; (2) “over-lysing” of the sample such that nuclei of the WBCs are stained and interfere with the nRBC count; (3) the existence of fragile lymphocytes, such that WBCs are unexpectedly hyper-sensitive to a lysing reagent (giving false positives); and/or (4) the existence of lyse-resistant nRBCs, such that the nRBCs are unexpectedly insensitive to a lysing reagent (giving false negatives). In practice, over-lysing or under-lysing is common on account of the variation in membrane rigidity of blood cells among samples of blood. As such, dependence on known light scatter and/or fluorescence emission detection techniques may result in an inaccurate and unreliable analysis for nRBCs, thereby preventing correct diagnoses and treatment for critically ill patients.